Gene Organization and Transcription Analysis of the Agrobacterium tumefaciens Glycogen ( glg ) Operon: Two Transcripts for the Single Phosphoglucomutase Gene

Abstract

ABSTRACT The gene organization and transcription of the Agrobacterium glg operon differ from those in other bacteria. Agrobacterium tumefaciens A348 contains a 9.1-kb gene cluster harboring genes for glycogen metabolism. The nucleotide sequence and gene organization of a region containing ADP-glucose pyrophosphorylase ( glgC ), glycogen synthetase ( glgA ), and phosphoglucomutase ( pgm ) genes have been previously described (A. Uttaro and R. A. Ugalde, Gene 150:117–122, 1994). In this work we report that the glycogen phosphorylase ( glgP ) and branching enzyme ( glgB ) genes are located immediately upstream of this region. The complete nucleotide sequences of the glgP and glgB genes were obtained, and mutants were constructed by targeted insertional mutagenesis with a kanamycin cassette. Enzymatic assays and reverse transcription PCR carried out with the wild type and with glgP and glgB mutants, as well as primer extension experiments and β-galactosidase fusions, revealed that this region containing five open reading frames ( glgPBCA and pgm ) is transcribed unidirectionally as a single operon under the control of a promoter located upstream of the glycogen phosphorylase gene ( glgP ). An alternative transcript was identified starting 168 bp upstream of an internal ATG start codon of the pgm gene, which is translated as a 71-amino-acid-shorter Pgm protein which complements in vivo a pgm mutant. This alternative transcript has a promoter with the motif TATCAAN 5 G, identified in octopine Ti plasmid as an autoinducible TraR promoter. This promoter is >200 times more efficient in A. tumefaciens than in Escherichia coli , as judged by the level of enzymatic activity of a lacZ-pgm fusion.